Intracytoplasmic sperm injection (ICSI) has increased the potential of the assisted reproductive technologies to propagate mammalian species and has provided an opportunity for research into cell cycle control and the mechanisms involved in sperm-induced oocyte activation. We have investigated the efficacy of ICSI in the rhesus monkey, the mechanism of fertilization following sperm injection and the cytoskeletal rearrangement that occurs upon oocyte activation. These studies were conducted on mature and to a lesser extent immature oocytes. Ejaculated sperm, washed, capacitated and activated before immobilization were injected into oocytes using conventional ICSI methodology. Sperm injection into mature oocytes induced oocyte activation (19/86%) and pronuclear formation. In contrast, sham injected oocytes did not activate readily (2/16;13%). To localize oocyte activation factor(s), sperm were separated mechanically into heads and tails and injected individually into mature oocytes. Activation occurred in 87% (20/23) of oocytes receiving heads. After tail injection, a single microtubule aster was nucleated and one pronucleus (PN) was seen in 4 of 21 oocytes. Intracytoplasmic injection of sperm extract (SE) resulted in oocyte activation at a significantly higher rate than that of sham injection (76% versus 13%). Sperm-induced oocyte activation was also evaluated in immature metaphase (MI) oocytes; activation occurred in 46% (12/26) of cases; however, only 8% of the activated oocytes exhibited 2 PN. Finally, -tubulin localization in untreated and taxol-treated oocytes was established as a marker for cytoplasmic changes associated with oocyte activation. These results are consistent with the hypothesis that sperm contain an oocyte activating factor(s) and the factor(s) is primarily localized in the sperm head. Moreover, an activation response is limited to mature oocytes and is accompanied by cytoskeletal changes analogous to those seen following conventional fertilization.